RESEARCH ARTICLE Cabbage stem flea beetle's (Psylliodes chrysocephala L.) susceptibility to pyrethroids and tolerance to thiacloprid in the Czech Republic ☯ ☯ Jitka Stara ID *, FrantisÏek Kocourek Department of Integrated Crop Protection against Insect Pests, Division of Crop Protection and Plant Health, Crop Research Institute, Prague, Czech Republic ☯ These authors contributed equally to this work. a1111111111 * [email protected] a1111111111 a1111111111 a1111111111 Abstract a1111111111 The cabbage stem flea beetle (CSFB), Psylliodes chrysocephala (Coleoptera: Chrysomeli- dae), has recently become a major pest species in winter oilseed rape in the Czech Repub- lic. The susceptibility of CSFB populations from two localities to six pyrethroids, two OPEN ACCESS neonicotinoids, one organophosphate and one oxadiazine was evaluated in 2015±2018 in glass vial experiments. The susceptibility of CSFB to thiacloprid and thiamethoxam was Citation: Stara J, Kocourek F (2019) Cabbage stem flea beetle's (Psylliodes chrysocephala L.) evaluated in feeding experiment in 2017 and 2018. High susceptibility of CSFB populations susceptibility to pyrethroids and tolerance to to lambda-cyhalothrin, cypermethrin, esfenvalerate, tau-fluvalinate, etofenprox, deltame- thiacloprid in the Czech Republic. PLoS ONE 14(9): thrin, chlorpyrifos, indoxacarb and acetamiprid was observed in the glass vial experiments. e0214702. https://doi.org/10.1371/journal. pone.0214702 The LC50 and LC90 data obtained for pyrethroids in these experiments in 2015 represent baseline for CSFB resistance monitoring to pyrethroids in the Czech Republic. High toler- Editor: Carla A Ng, University of Pittsburgh, UNITED STATES ance of CSFB to thiacloprid of CSFB was demonstrated in glass vial and the feeding experi- ment, too. Received: March 18, 2019 Accepted: September 4, 2019 Published: September 20, 2019 Copyright: © 2019 StaraÂ, Kocourek. This is an open access article distributed under the terms of the Introduction Creative Commons Attribution License, which permits unrestricted use, distribution, and The cabbage stem flea beetle (CSFB), Psylliodes chrysocephala Linnaeus 1758 (Coleoptera: reproduction in any medium, provided the original Chrysomelidae) is one of the major pest species of winter oilseed rape in Western Europe author and source are credited. [1,2,3]. CSFB has one generation per year and undergoes summer diapause. The period of Data Availability Statement: All relevant data are adult summer diapause is not influenced through constant or changing environmental tem- within the manuscript. peratures or short or long daylight conditions [4]. In autumn, adults migrate into new fields of Funding: The work was financially supported oilseed rape. Females lay eggs after mating on or close to the plants. After hatching, the larvae through a grant from the projects MZE-RO0418 feed on leaf bases and terminal leaflets. All the developmental stages can overwinter and and NAZV QK 1820081 of the ministry of females can resume laying eggs in the spring. Agriculture of the Czech Republic. Since 2009, the first evidence of CSFB resistance to pyrethroids has been demonstrated as a Competing interests: The authors have declared consequence of the long-term selective pressure of pyrethroids applied for the control of CSFB that no competing interests exist. in oilseed rape [2]. The resistance factor according to the LC50 values for lambda-cyhalothrin PLOS ONE | https://doi.org/10.1371/journal.pone.0214702 September 20, 2019 1 / 12 Cabbage stem flea beetle's (Psylliodes chrysocephala L.) susceptibility and tolerance reached 80 when 28 local populations of CSFB from Germany were examined [1]. Failure to control CSFB has become a major issue in Germany and the United Kingdom [3]. Until now, no data were available about the susceptibility of CSFB to insecticides in the Czech Republic. Since 2013, three active neonicotinoid ingredients, thiamethoxam, clothianidin and imida- cloprid, have been restricted in Europe for seed treatments [5]. Until the start of the restriction, CSFB control in northern Germany and further regions of Western Europe has relied on seed dressings with neonicotinoids or spraying with pyrethroids in autumn [2,6]. Similarly, CSFB in the Czech Republic was effectively controlled until 2013 by seed dressings of winter oilseed rape with neonicotinoids. Using pyrethroids in oilseed rape in autumn was sporadic. In 2014, the first economically important injuries to oilseed rape caused by CSFB were recorded by farmers in the central part of the Czech Republic. Since 2015, CSFB has been controlled in most of the regions of the Czech Republic by spraying with pyrethroids. The present study brings the results of i) evaluation of the susceptibility of local populations of CSFB from the Czech Republic to six pyrethroids, two neonicotinoids, one organophoshate and one oxadiazine in glass vial experiment, ii) evaluation of susceptibility of CSFB to two neo- nicotinoids in feeding experiments. Materials and methods Sample collection The field populations of CSFB originated from winter oilseed rape fields located in PoteÏhy (49Ê5209.695@N, 15Ê25012.344@E) and Prague (50Ê5015.437@N, 14Ê17058.980@E). CSFB adults were collected by sweeping during June in 2015±2018. According to the CSFB population den- sity, 500±1,500 adults per population were collected in particular years and populations. Bee- tles with some oilseed rape plant material in the net insulators were transported to the laboratory. Prior to initiating the bioassays, samples of the beetles were stored at +5ÊC. The transported and stored beetles were fed winter oilseed rape leaves. The beetles were tested for susceptibility within 48 h after collection. Only live, mobile insects were used for the bioassays. A total of 4,876 CSFB beetles were tested in bioassays in 2015±2018. Bioassays Insecticides. Six analytical standards (lambda-cyhalothrin, cypermethrin, esfenvalerate, tau-fluvalinate, etofenprox and deltamethrin), one organophosphate (chlorpyrifos), one oxa- diazine (indoxacarb) and two neonicotinoids (thiacloprid and acetamiprid) were obtained from Sigma Aldrich Co. LLC., Prague, Czech Republic. Four to seven doses of insecticides diluted with acetone were used to generate dose-response curves. The rates recommended in the Czech Republic for the field application were calculated assuming that the highest recom- mended rate of a commercial product in 400 L of water is used per hectare (Table 1). To gener- ate dose-response curves, the rates of the tested insecticides corresponding to 0.1%-1,000% of the field rates were tested (Table 1). Thiacloprid was tested in 2015±2017 as the analytical stan- dard and in 2017±2018 as the formulated product Biscaya 240 OD. Stock solutions of Biscaya 240 OD were prepared by dissolving 140.4 mg of Biscaya 240 OD formulation (containing 32.4 mg of thiacloprid) in 2 mL of distilled water, and subsequently adjusted to 100 mL with acetone. All further dilutions were made in acetone. Glass vial experiment. IRAC adult vial tests [7] no. 31 (pyrethroids), no. 21 (neonicoti- noids), no. 25 (organophosphates) and no. 27 (oxadiazines) were used to conduct the bioassay. The glass vials (P-lab, CZ, internal surface 32.4 cm2) were filled with 520 μL of insecticide and rotated on a hotdog roller at room temperature until the acetone was completely evaporated. Subsequently, the beetles were transferred into each vial. Three replicates were used, and 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0214702 September 20, 2019 2 / 12 Cabbage stem flea beetle's (Psylliodes chrysocephala L.) susceptibility and tolerance Table 1. The recommended field rates and tested rates of insecticides used to evaluate CSFB susceptibility from the Prague and PoteÏhy localities in 2015±2018. Tested insecticides Field rates (μg/cm2) Tested rates (μg/cm2) cypermethrin 0.25 0.0025, 0.025, 0.05, 0.25 deltamethrin 0.075 0.000075, 0.00075, 0.0075, 0.015, 0.075, 0.75 esfenvalerate 0.075 0.0075, 0.015, 0.075, 0.75 etofenprox 0.6 0.06, 0.12, 0.60, 6.00 lambda-cyhalothrin 0.075 0.00075, 0.0075, 0.015, 0.075, 0.75 tau-fluvalinate 0.48 0.00048, 0.0048, 0.0192, 0.048, 0.096, 0.48, 4.80 chlorpyrifos 3.00 0.03, 0.0752, 0.15, 0.30, 0.60, 3.00 indoxacarb 0.255 0.024, 0.048, 0.24, 2.40 acetamiprid 0.20 0.002, 0.02, 0.04, 0.20 thiacloprid 0.72 0.072, 0.144, 0.72, 1.44, 7.20 https://doi.org/10.1371/journal.pone.0214702.t001 beetles were used for each insecticide dose in each replication. The control vials were treated with pure acetone. The vials were incubated under controlled conditions at 20ÊC and 60% rela- tive humidity with a 16:8 h light:dark photoperiod. The number of severely affected beetles (i.e., dead and moribund) was counted after 48 h. Feeding experiment. Oilseed rape of the Exssence variety was sown at the rate of 400,000 seeds per ha with a plot seeder Wintersteiger (Wintersteiger SaÈgen GmbH, Arnstadt, Ger- many) on small plots (0.2 ha) in the field in Prague locality with three variants: (1) without seed dressing, (2) seed dressing with Sonido (thiacloprid), and (3) seed dressing with Cruiser (thiamethoxam). The plots were left without treatment with herbicides or insecticides. The conditions of the experiment permitted the natural infestation by CSFB adults. The incidences of adults in the plots were monitored by Moericke yellow traps. The traps were inspected in 2±3 day intervals. In 2017, the plants from the experiment were collected in two periods, 24 days and 35 days after sowing of the oilseed rape seeds. 5.5 and 6.2 adults were caught per trap from the plant emergence to the first and second term of plant collection, respectively. Growth stage of oilseed rape plats according to BBCH-scale corresponding to dates of plant sampling are presented in Table 2. In 2018, the plants were collected 29 days after sowing of the oilseed Table 2. Content of pesticide residues in oilseed rape plants (values ± standard deviation), average weight of plants before analysis and average injury to plants caused by CSFB.